Novel Requirements for Akt1 in T Cell Commitment

摘要 Akt1、Akt2和Akt3是丝氨酸/苏氨酸激酶家族的成员，其是PI3K的关键效应子。的 Akt家族已被充分表征为促进细胞存活、葡萄糖代谢和增殖。 包括发育中的T细胞在内的多种细胞类型。胸腺细胞表达不同程度的所有Akt亚型。 单个或多个Akt基因的消融导致T细胞发育的差异阻断。三个都丢了 Akt同种型导致早期发育缺陷，原因是CD4-CD8-双 阴性（DN）胸腺细胞。单独表达Akt2或Akt3足以维持DN存活;然而， DN3分化和DN4胸腺细胞增殖沿着细胞存活， 发育阶段这些不同的表型表明，不同的Akt亚型调节不同的 或者需要不同剂量的Akt活性来促进阶段特异性细胞存活， 增殖和分化。由于目前没有异构体特异性功能的证据，我们将重点放在了 注意表征调节Akt活性剂量的机制。表面受体，包括 （前）T细胞受体和细胞因子受体激活PI3K以产生PIP3。PIP3与 Akt的Pleckstrin同源（PH）结构域需要将Akt募集到质膜， activation.因此，精细控制PI3K活性和PIP3可用性可以直接调节Akt活化水平。 该应用解决了用于间接控制Akt的日益重要的替代机制 活性：通过产生IP4，一种结构上类似于PIP3的可溶性肌醇多磷酸。IP4是 在（前）TCR刺激后产生，并与PIP3竞争结合Akt PH结构域。 IP4缺陷的胸腺细胞在DN3和DN4表达前TCR后过度激活Akt 阶段引人注目的是，Akt过度活跃导致加速的β选择，过早重编程为糖酵解， 代谢和Notch依赖性的丧失。这使我们提出了以下总体假设：1）preTCR- 诱导的IP4产生限制Akt依赖性代谢重编程和增殖， 足够的Notch信号传导和2）preTCR和Notch协同信号传导是T细胞谱系所需的 规范.这项研究的成功完成将改变我们目前对T细胞系的认识 通过揭示1）在施加Notch检查点中对preTCR的意想不到的要求，以及 共刺激Notch功能，2）控制DN胸腺细胞的新的preTCR反馈机制 通过调节Akt活性和代谢重编程的增殖/成熟;和3）非典型的 活化TCF 1依赖性转录以指定T细胞定型的途径。

ABSTRACT Akt1, Akt2 and Akt3 are members of a family of serine/threonine kinases that are key effectors of PI3K. The Akt family has been well characterized to promote cell survival, glucose metabolism and proliferation in many cell types including developing T cells. Thymocytes express all Akt isoforms to varying degrees. Ablation of single or multiple Akt genes results in differential blocks in T cell development. Loss of all three Akt isoforms leads to an early developmental defect due to decreased survival of CD4–CD8– double negative (DN) thymocytes. Expression of Akt2 or Akt3 alone is sufficient to preserve DN survival; however, DN3 differentiation and DN4 thymocyte proliferation remain defective along with survival of cells at later developmental stages. These distinct phenotypes suggest either that different Akt isoforms regulate distinct functions or that different doses of Akt activity are required to promote stage-specific cell survival, proliferation and differentiation. With no current evidence for isoform-specific functions, we focused our attention on characterizing mechanisms for regulating the dose of Akt activity. Surface receptors, including (pre-)T cell receptor and cytokine receptors, activate PI3K to generate PIP3. Interactions between PIP3 and the Pleckstrin homology (PH) domain of Akt are required to recruit Akt to the plasma membrane for activation. Thus, fine control of PI3K activity and PIP3 availability can directly regulate Akt activation levels. This application addresses an increasingly important alternative mechanism for indirectly controlling Akt activity: via generation of IP4, a soluble inositol polyphosphate that is structurally similar to PIP3. IP4 is generated following (pre)TCR stimulation and competes with PIP3 for binding to the Akt PH domain. Thymocytes deficient in IP4 activate Akt excessively following expression of preTCR at the DN3 and DN4 stages. Strikingly, Akt hyperactivity leads to accelerated β selection, premature reprogramming to glycolytic metabolism and loss of Notch dependency. This leads us to propose the global hypotheses that 1) preTCR- induced IP4 generation restricts Akt-dependent metabolic reprogramming and proliferation to ensure adequate Notch signaling and 2) preTCR and Notch cooperative signaling is required for T cell lineage specification. Successful completion of this study will shift our current understanding of T lineage specification by revealing 1) an unexpected requirement for preTCR in imposing a Notch checkpoint and in co-stimulating Notch function, 2) a novel preTCR feedback mechanism that controls DN thymocyte proliferation/maturation via tuning of Akt activity and metabolic reprogramming; and 3) a non-canonical pathway for activating TCF1-dependent transcription to specify T cell commitment.